There is a continuing need in medical practice and research, and in analytical and diagnostic procedures, for rapid and accurate determinations of chemical and biological substances present in various fluids, such as biological fluids. For example, the presence of drugs, narcotics, hormones, proteins, toxins, microorganisms, viruses, steroids or nucleic acids must be rapidly and accurately detected for effective research, diagnosis or treatment of various diseases or conditions.
A wide variety of analytical methods have been developed in recent decades to detect chemical or biological substances. Most of such methods rely on what are known in the art as "specific binding" reactions in which an unknown substance to be detected (known as a "specific binding ligand") reacts specifically and preferentially with a corresponding "receptor" molecule. Most well known specific binding reactions occur between immunoreactants, such as antibodies and antigens, but other specific binding reactions (such as avidin with biotin, a sugar with a lectin or hormone with a receptor) are also known.
Many of the assay formats known in the art require one or more of the reactants to be immobilized on a solid substrate so that nonimmobilized reactants can be separated from immobilized reactants. Assays which require the presence of a solid phase for separation are generally known as "heterogeneous" and usually require a wash step.
In different assay formats which are known as "homogeneous", no separation step is generally needed because the desired signal can be detected in solution in the presence of all reactants. The elimination of wash or separation steps can be quite important in certain situations, such as in the use of dry analytical elements in automated equipment. Homogeneous assays have been commercialized and described in considerable literature, notably by Syva Corporation (for example, U.S. Pat. No. 3,935,074 of Rubenstein et al). In most homogeneous assays, an enzyme label is modulated in some fashion so that enzyme activity can be correlated to the amount of target specific binding ligand in a sample.
In homogeneous assays developed by Syva Corporation (EMIT.TM. assays), anti-ligand antibody partitions between free ligand and a conjugate of ligand and enzyme label. Anti-ligand antibody which binds to the conjugate reduces the catalytic activity of the enzyme. Despite its advantages, such assays have a primary limitation which is in the preparation of the conjugate. The enzyme commonly used in such assays (glucose dehydrogenase) has numerous substitution sites that are equally accessible, but only one of those sites is critical for the modulation of enzyme activity by the anti-ligand antibody. Thus, high substitution ratios are required to ensure that the majority of enzyme molecules are susceptible to inhibition. Different ligands alter the behavior of the resulting conjugate in terms of its solubility, retention of enzyme activity and susceptibility to inhibition. Variations in the conjugate may result from the manufacturing procedures, and thereby provide another cause for erratic assay performance. In other words, the known homogeneous assays are sensitive to small variations in concentration of the enzyme-ligand conjugate. In addition, the choice of enzyme labels is limited.
Another type of homogeneous assay, [Henderson et al, Clin. Chem., 32(9), pages 1637-1641, (1986)], uses the assembly of a functional reporter enzyme (for example, .beta.-galactosidase) from a pair of enzyme fragments, one of which is conjugated with target ligand. Anti-ligand antibody then interferes with assembly of the reporter enzyme by binding to the conjugate in the absence of target ligand. Two features make this assay difficult to carry out. The derivatization of the smaller enzyme fragment with target ligand can alter the assembly of functional enzyme or yield a conjugated fragment whose incorporation into functional enzyme is not blocked by anti-ligand antibody. Secondly, the equilibrium in the system of ligand, anti-ligand antibody and enzyme fragment conjugate lies near fully assembled enzyme and the resulting immunoassay is only transiently ligand dependent. That is, at equilibrium, the .beta.-galactosidase will be fully active, independent of analyte. In other words, the assay is not kinetically robust.
There remains a need for an assay which is kinetically robust and uses reactants which are simple to manufacture with little variability. It would also be desirable to avoid separation steps which are common in heterogeneous assays.